The present invention relates to methods and systems for spectroscopy, and more particularly, to Raman Spectroscopy.
The wavelength of light scattered inelastically from molecular species can be shifted due to energy coupling to the internal vibrational modes of the scattering species. Such frequency shifts may result in a discrete array of sharp lines corresponding to harmonic modes of the molecule. This spectral shift is generally referred to as the “Stokes” shift or the “Raman” effect. Raman spectroscopy may be used to identify the presence of a target molecule, to analyze the structure and/or densities of molecular species, and/or for other purposes. However, fluorescence produced by elastic scattering and/or stray light may obscure the Raman portion of the spectrum.
Shifted Excitation Raman Difference Spectroscopy (“SERDS”) may be used to reduce interference from fluorescence and stray light by shifting the frequency of a laser light that is impinged on a specimen. The Raman bands are generally shifted in response to a shift in excitation frequency, and the broad background fluorescence and stray light are generally much less affected by the excitation frequency shift. SERDS generally involves a subtraction of two spectra obtained from two different (shifted) excitation frequencies. The subtraction can result in a derivative spectrum that may reduce the background and fluorescence spectra. See S. E. J. Bell, E. S. O. Bourguignon, and A. Dennis, “Analysis of luminescent samples using subtracted shifted raman spectroscopy,” Analyst, vol. 123, no. 8, 1729–1734 (1998); P. Matousek, M. Towrie, and A. W. Parker, “Fluorescence background suppression in raman spectroscopy using combined kerr gated and shifted excitation raman difference techniques,” Journal of Raman Spectroscopy, vol. 33, no. 4, 238–242 (2002); A. P. Shreve, N. J. Cherepy, and R. A. Mathies, “Effective rejection of fluorescence interference in raman-spectroscopy using a shifted excitation difference technique,” Applied Spectrocopy, vol. 46, no. 4, 707–711 (1992); J. Zhao, M. M. Carrabba, and F. S. Allen, “Automated fluorescence rejection using shifted excitation raman difference spectroscopy,” Applied Spectroscopy, vol. 56, no. 7,934–945 (2002).
However, SERDS may not sufficiently reduce fluorescence and stray light in all environments. For example, Raman spectroscopy may be more difficult in diffuse media, such as biological tissue, because the Raman effect may be obscured by a high level of incoherent fluorescence spectra and/or by the diversity of molecules and the associated Raman signals that may be present in the media. Raman spectra may also be difficult to isolate in diffuse media because of the spatial incoherence of the scattered signal.